Molecular pharmacology
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Molecular pharmacology · Dec 2004
Viral macrophage inflammatory protein-II and fractalkine (CX3CL1) chimeras identify molecular determinants of affinity, efficacy, and selectivity at CX3CR1.
Fractalkine (FKN/CX3CL1) is a cell surface-expressed chemokine involved in many aspects of leukocyte trafficking and activation. The various structural domains of FKN play distinct roles in its ability to bind and activate its receptor, CX3CR1. A human herpesvirus 8-encoded chemokine, termed viral macrophage inflammatory protein (vMIP)-II, is structurally similar to FKN; vMIP-II is a nonselective chemokine receptor antagonist (binding multiple chemokine receptors, including CX3CR1). ⋯ Substitution of the vMIP-II N terminus with that of FKN created an agonist that was just as potent and efficacious as FKN for binding and stimulating CX3CR1, whereas replacement of the FKN N terminus with the cognate domain of vMIP-II disrupted the ability of FKN to bind CX3CR1. Furthermore, the entire N terminus of FKN was necessary for the high-affinity and full agonist properties of FKN at CX3CR1. These results refine the pharmacophore for chemokine binding to and activation of CX3CR1 and demonstrate the usefulness of modified virally encoded chemokines as templates for the development of selective chemokine receptor antagonists.
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Molecular pharmacology · Dec 2004
Critical role of reactive oxygen species and mitochondrial membrane potential in Korean mistletoe lectin-induced apoptosis in human hepatocarcinoma cells.
Viscum album L. coloratum agglutinin (VCA), isolated from Korean mistletoe, is a strong inducer of apoptosis in a variety of tumor cells; however, the underlying molecular mechanisms responsible are not clear. Here, we show that VCA induces apoptotic killing, as demonstrated by DNA fragmentation, Hoechst 33258 staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and flow cytometry analysis in hepatocarcinoma Hep3B cells. VCA treatment results in a significant increase in reactive oxygen species (ROS) and loss of mitochondrial membrane potential (DeltaPsim). ⋯ Moreover, Hep3B cells overexpressing JNK1 or stress-activated protein kinase kinase (SEK1) seem to be more susceptible to cell death from ROS and loss of DeltaPsim induced by VCA, whereas expression of dominant-negative JNK1 or SEK1 in Hep3B cells do not. These data suggest that JNK phosphorylation may be a major regulator involved in VCA-induced apoptosis. Together, these results suggest that VCA induces apoptosis by inducing ROS production and a loss of DeltaPsim, in which JNK phosphorylation plays a critical role in these events.
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Molecular pharmacology · Nov 2004
5beta-reduced neuroactive steroids are novel voltage-dependent blockers of T-type Ca2+ channels in rat sensory neurons in vitro and potent peripheral analgesics in vivo.
T-type Ca(2+) channels are believed to play an important role in pain perception, and anesthetic steroids such as alphaxalone and allopregnanolone, which have a 5alpha-configuration at the steroid A, B ring fusion, are known to inhibit T-type Ca(2+) channels and cause analgesia in a thermal nociceptive model (Soc Neurosci Abstr 29:657.9, 2003). To define further the structure-activity relationships for steroid analgesia, we synthesized and examined a series of 5beta-reduced steroids for their ability to induce thermal antinociception in rats when injected locally into the peripheral receptive fields of the nociceptors and studied their effects on T-type Ca(2+) channel function in vitro. We found that most of the steroids completely blocked T-type Ca(2+) currents in vitro with IC(50) values at a holding potential of -90 mV ranging from 2.8 to 40 microM. ⋯ For the most potent steroids, we found that other voltage-gated currents were not significantly affected at concentrations that produce nearly maximal blockade of T currents. All tested compounds induced dose-dependent analgesia in thermal nociceptive testing; the most potent effect (ED(50), 30 ng/100 microl) obtained with a compound [(3beta,5beta,17beta)-3-hydroxyandrostane-17-carbonitrile] that was also the most effective blocker of T currents. Compared with previously studied 5alpha-reduced steroids, these 5beta-reduced steroids are more efficacious blockers of neuronal T-type Ca(2+) channels and are potentially useful as new experimental reagents for understanding the role of neuronal T-type Ca(2+) channels in peripheral pain pathways.
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Molecular pharmacology · Sep 2004
Acetaminophen: a central analgesic drug that involves a spinal tropisetron-sensitive, non-5-HT(3) receptor-mediated effect.
The reversal of the antinociceptive effect of systemically administered acetaminophen (paracetamol) by intrathecal administration of the potent 5-HT(3) receptor antagonist tropisetron has been reported in rats subjected to the paw pressure test, suggesting that acetaminophen action is mediated through spinal 5-HT(3) receptors. However, more recent data, showing differences between the pharmacological profiles of various 5-HT(3) receptor antagonists, led us to reconsider the involvement of spinal 5-HT(3) receptors. To address this question, two different approaches were used: 1) electrophysiological recordings to assess whether acetaminophen directly modulates 5-HT(3) receptor activity and 2) pharmacological investigations with various 5-HT(3) receptor antagonists and spinal 5-HT(3) receptors antisense oligodeoxynucleotides (AODNs) to determine how those treatments might affect the antinociceptive action of acetaminophen. ⋯ Unlike tropisetron, other 5-HT(3) receptor antagonists, such as ondansetron and granisetron, injected intrathecally were unable to reverse the antinociceptive effect of acetaminophen. Moreover, pretreatment with AODNs did not reverse the acetaminophen-induced antinociceptive effect, although it suppressed the antinociceptive effect of m-chlorophenylbiguanide, a specific agonist of 5-HT(3) receptors, and significantly reduced (30%) the expression of these receptors in the dorsal horn of the spinal cord. These results suggest that acetaminophen-induced antinociceptive action involves a spinal tropisetron-sensitive receptor that is not the 5-HT(3) receptor and that remains to be identified.
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Molecular pharmacology · Sep 2004
A vascular endothelial growth factor receptor-2 kinase inhibitor potentiates the activity of the conventional chemotherapeutic agents paclitaxel and doxorubicin in tumor xenograft models.
Inhibition of angiogenesis may have wide use in the treatment of cancer; however, this approach alone will not cause tumor regression but may only slow the growth of solid tumors. The clinical potential of antiangiogenic agents may be increased by combining them with conventional chemotherapeutics. 4-[4-(1-Amino-1-methylethyl)phenyl]-2-[4-(2-morpholin-4-yl-ethyl)phenylamino]pyrimidine-5-carbonitrile (JNJ-17029259) represents a novel structural class of 5-cyanopyrimidines that are orally available, selective, nanomolar inhibitors of the vascular endothelial growth factor receptor-2 (VEGF-R2) and other tyrosine kinases involved in angiogenesis, such as platelet-derived growth factor receptor, fibroblast growth factor receptor, VEGF-R1, and VEGF-R3, but have little activity on other kinase families. At nanomolar levels, JNJ-17029259 blocks VEGF-stimulated mitogen-activated protein kinase signaling, proliferation/migration, and VEGF-R2 phosphorylation in human endothelial cells; inhibits the formation of vascular sprouting in the rat aortic ring model of angiogenesis; and interferes with the development of new veins and arteries in the chorioallantoic membrane assay. ⋯ Histological examination revealed that the tumors have evidence of reduced vascularity after treatment. In addition, JNJ-17029259 enhances the effects of the conventional chemotherapeutic drugs doxorubicin and paclitaxel in xenograft models when administered orally in combination therapy. An orally available angiogenesis inhibitor that can be used in conjunction with standard chemotherapeutic agents to augment their activity may have therapeutic benefit in stopping the progression of cancer and preventing metastasis.